Project Summary/Abstract The overarching goal of our research is to understand the molecular basis of disease caused by enteric bacterial pathogens, and to leverage this knowledge to develop therapeutic agents that will benefit the Veteran population. A major focus of our laboratory is the study of the bacterial pathogen, Clostridium difficile, which causes healthcare-associated infections, and has been designated an ?Urgent Threat? by the Centers for Disease Control and Prevention. The risk factors for acquiring C. difficile infection include age over 65 years, antibiotic treatment, and use of acid-suppressors; this combination of risk factors makes Veterans especially vulnerable to infection. Our ongoing epidemiological studies show that C. difficile infection rates in the Tucson VA (and other Arizona) hospitals are above the national average, and that many patients harbor `hyper-virulent' epidemic-associated strains of the pathogen. We have characterized VA C. difficile strains in detail, uncovered novel virulence strategies employed by these organisms, used cutting-edge genome analyses to track their evolution and spread, and provided feedback to physicians in an effort to reduce incidence of infections. We have also made several innovative advances in exploring the molecular mechanisms by which C. difficile causes disease. For these efforts, we have extensively used mass spectrometry techniques to globally analyze various strains to discern their unique virulence potential. Further, we have adopted, and greatly improved, recent methodologies to genetically manipulate C. difficile in order to understand its virulence; this has allowed us to knock out, or limit the expression of, various genes. Beyond the pioneering work revealing that C. difficile toxins contribute to disease, we have uncovered key roles for non-toxin factors including flagella, capsular polysaccharides and superoxide reductase in virulence. We have also used mass spectrometry to understand corresponding changes in host cells. Finally, building on our understanding of how C. difficile attaches to the host intestine, we recently designed and engineered a novel, safe ?synthetic biologic? agent to express C. difficile adhesins. In an animal model of infection, the agent was able to completely block C. difficile infection and disease. This product, the first generation of which is fully supported by our current VA Merit award, is now protected by a provisional patent, and is currently under intensive further refinement. It is anticipated that the agent will be an effective, safe, ?first-in-kind? non-antibiotic option to prevent C. difficile infection in Veterans, as well as all susceptible individuals.